WO2023009099A1 - Kit de pièces, procédé et système pour pcr - Google Patents

Kit de pièces, procédé et système pour pcr Download PDF

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Publication number
WO2023009099A1
WO2023009099A1 PCT/US2021/043118 US2021043118W WO2023009099A1 WO 2023009099 A1 WO2023009099 A1 WO 2023009099A1 US 2021043118 W US2021043118 W US 2021043118W WO 2023009099 A1 WO2023009099 A1 WO 2023009099A1
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Prior art keywords
oligonucleotide
bead
nucleic acid
thermocycling
bound
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PCT/US2021/043118
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English (en)
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Viktor Shkolnikov
Alexander Govyadinov
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Hewlett-Packard Development Company, L.P.
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Priority to PCT/US2021/043118 priority Critical patent/WO2023009099A1/fr
Publication of WO2023009099A1 publication Critical patent/WO2023009099A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • PCR Polymerase Chain Reaction
  • pathogens for example bacteria or viruses
  • personalised medicine requires genotyping using PCR in which the detection of one or more biomarkers, for example specific mutations, may influence clinical decisions on the nature or type of medical intervention.
  • PCR subjects a sample to amplification conditions in the presence of an enzyme capable of elongating nucleic acid strands, for example a polymerase.
  • an enzyme capable of elongating nucleic acid strands for example a polymerase.
  • the three basic steps of a single round or cycle of PCR amplification are denaturation, annealing and chain extension, each optimally taking place at different temperatures (typically 94-98 °C for denaturation; 50-65 °C for annealing, and 70-80 °C for chain extension, depending on polymerase), with each set of three steps being known by the term “thermocycling”.
  • the amplification products (amplicons) are detected optically, typically using fluorescent reporters.
  • Figure 1 is a schematic of an example PCR kit-of-parts
  • Figure 2 shows a mechanism of cleavage of a photocleavable linker
  • Figure 3 is a schematic of an example PCR kit-of-parts;
  • Figure 4 shows an example microfluidic device for use in an example PCR system;
  • Figure 5 is a schematic of an example PCR method.
  • a weight range of approximately 1 wt.% to approximately 20 wt.% should be interpreted to include not only the explicitly recited concentration limits of 1 wt.% to approximately 20 wt.%, but also to include individual concentrations such as 2 wt.%, 3 wt.%, 4 wt.%, and sub- ranges such as 5 wt.% to 10wt.%, 10 wt.% to 20 wt.%, etc. [00013]
  • concentration limits such as 2 wt.%, 3 wt.%, 4 wt.%
  • sub- ranges such as 5 wt.% to 10wt.%, 10 wt.% to 20 wt.%, etc.
  • photocleavable linker refers to a reactive moiety which covalently couples or binds a molecule of interest to a solid-phase bead, and which undergoes photolysis under controlled conditions to cleave or release the molecule of interest from the bead.
  • thermalally cleavable linker refers to a moiety which non-co valently couples or binds a molecule of interest to a solid-phase bead at room temperature, and which dissociates from the molecule of interest at elevated temperatures to cleave or release the molecule of interest from the bead.
  • PCR Polymerase Chain Reaction
  • dNTP refers to the 2’-deoxynucleotide triphosphates used in PCR.
  • the four standard dNTPs are 2’- deoxyadenosine 5’-triphosphate, 2’-deoxyguanosine 5’-triphosphate, 2’-deoxycytosine 5’-triphosphate and thymidine 5’-triphosphate (already lacking a 2’-hydroxyl), though modified dNTPs, for example non-natural nucleotides incorporating labels or reporter molecules, or reactive moieties may also be used (typically in the form of nucleobase modifications such as C7-modified deaza-guanine orC7-modified deaza-adenine orC5- modified cytosine or C5-modified thymidine).
  • Non-natural nucleotides having for example sugar modifications such as 2’-F or 2’-OMe modifications, or “LNA”, having an O-CH2 linker between the 2’ and 4’ positions on the sugar
  • backbone modifications such as phosphorothioates, or phosphorothiolates
  • unnatural bases such as the pyrimidine analog 6-amino-5-nitro-3-(T-p-D-2'-deoxyribofuranosyl)- 2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(T-
  • P-D-2'-deoxyribofuranosyl)-imidazo[1 ,2-a]-1 ,3,5-triazin-4(8H)-one (dP).
  • the term “primer” refers to a short single stranded nucleic acid, typically an oligodeoxynucleotide (also referred to as an oligonucleotide herein), of about 15 to 30 nucleotides in length.
  • a primer is designed to base pair in a specific or complementary manner to a nucleic acid sequence of interest, and so is considered specific to that nucleic acid.
  • DNA is directional, with the 3’ end of one strand forming base pairs with the 5’-end of the counter strand and a primer is usually designed so that its 5’-end base pairs to the 3’-end of the nucleic acid of interest so that DNA synthesis (which occurs in a 5’ to 3’ direction) to elongate the primer can occur.
  • oligonucleotide pair refers to a set of two oligonucleotides that can serve as forward and reverse primers for a nucleic acid of interest.
  • each strand requires a primer: the forward primer attaches to the start codon of the template DNA strand (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand).
  • the 5'-end of each primer binds to the 3'-end of the complementary DNA strand of the nucleic acid of interest.
  • nucleic acid of interest refers to a polynucleotide sequence, typically of at least one hundred, two hundred, three hundred, four hundred, five hundred or up to one thousand nucleotides in length.
  • the polynucleotide sequence may be specific to a particular organism such as a pathogen, or may be suspected of having a particular mutation along its length, and will encode a particular polypeptide or protein, or mutant form thereof.
  • the polynucleotide sequence may encode the spike protein of SARS-CoV-2, or may encode a mutant form of the epidermal growth factor receptor (EGFR) the presence or absence of which renders a patient more or less likely to respond well to cancer treatments such as erlotinib or gefitinib.
  • EGFR epidermal growth factor receptor
  • PCR subjects a sample to multiple rounds of thermocycling in the presence of an enzyme capable of elongating nucleic acid strands, for example a polymerase.
  • an enzyme capable of elongating nucleic acid strands for example a polymerase.
  • Polymerases catalyse the reaction between a deoxynucleotide triphosphate and a DNA strand, producing an elongated DNA strand bearing one more nucleotide (from the deoxynucleotide triphosphate), and pyrophosphate as a by-product.
  • thermostable polymerases such as Taq polymerase (from Therm us aquaticus), Pfu polymerase (from Pyrococcus furiosus), and Bst polymerase (from Bacillus stearothermophilus) .
  • the DNA strand that is elongated in PCR is usually in the form of an oligonucleotide primer specific to a target nucleic acid sequence of interest, which is elongated using a mixture of deoxy ribonucleotide triphosphates (dNTPs).
  • dNTPs deoxy ribonucleotide triphosphates
  • dNTPs corresponding to the four nucleobases found in DNA (adenine, guanosine, thymine and cytosine) are required: 2’- deoxyadenosine 5’-triphosphate, 2’-deoxyguanosine 5’-triphosphate, 2’-deoxycytosine 5’-triphosphate and thymidine 5’-triphosphate.
  • the three basic steps of a single round or cycle of PCR amplification are denaturation, annealing and chain extension, each optimally taking place at different temperatures (typically 94-98 °C for denaturation; 50-65 °C forannealing, and 70-80 °C for chain extension, depending on polymerase), hence the term thermocycling.
  • the denaturation step separates the two strands of double-stranded DNA, with each strand acting as a template in the later chain extension step in which a complete complementary strand to the template is produced.
  • An oligonucleotide primer (typically comprising 15 to 30 nucleotides to ensure a balance of good specificity and efficient hybridization) is annealed to the 3’-end of each single stranded DNA molecule, and acts as a template for the synthesis of the new strand.
  • a DNA polymerase, and a mix of dNTPs then synthesize the new strand in the chain extension step, using the original single strand of DNA as its template. Since both strands of the original DNA duplex are used as templates, a singe round or cycle of PCR results in a doubling of the number of DNA duplexes. The number of copies thus increases exponentially with the number of cycles of amplification: after 2 cycles, four DNA duplexes are present in the sample, while after 3 cycles, 8 duplexes are present.
  • PCR is typically done on a prepared sample of 10-50 pl_ and quantified by monitoring the fluorescence of the fluid as it is thermally cycled. Since the fluorescence is proportional to the amount of nucleic acid (double stranded DNA), the fluorescence intensity increases as the number of cycles of amplification (the amount of double stranded DNA produced) increases. However, in order to achieve a high enough signal to noise ratio, typically 40 cycles are required.
  • the amplification products are detected optically, typically using fluorescent reporters.
  • Fluorescent reporter molecules used in PCR include non-specific fluorescent dyes, such as SYBR Green I, which has a distinct emission spectrum when intercalated into any double-stranded DNA, leading to an increase in fluorescence as more double-stranded DNA is produced.
  • Other suitable reporter molecules include target-specific fluorescent reporter molecules, such as the TaqMan hydrolysis probes of target-specific nucleic acids labelled with fluorescent reporter and quencher, with the probe being hydrolyzed by the exonuclease activity of the Taq polymerase, releasing the reporter from the quencher and again leading to an increase in fluorescence.
  • Reporter molecules may also be linked to a primer to be used in the PCR amplification, such as in the Scorpion® system, a single-stranded bi-labeled fluorescent probe sequence forming a hairpin-loop conformation with a 5’ end reporter and an internal quencher directly linked to the 5’ end of a primer via a blocker (which prevents the polymerase from extending the primer).
  • the polymerase extends the primer and synthesizes the complementary strand of the specific target sequence.
  • the hairpin-loop unfolds and the loop-region ofthe probe hybridizes intramolecularly to the newly synthesized target sequence. Now that the reporter is no longer in close proximity to the quencher, fluorescence emission may take place.
  • the fluorescent signal is detected and is directly proportional to the amount of amplified nucleic acid.
  • reporter dyes have different, and distinct emission spectra, combinations of different reporters can be strategically used in multiplex reactions.
  • SYBR Green I other cyanine dyes such as Cy3, or Cy5 can be used, as well as rhodamine dyes. Cy3 has a fluorescence emission at 570 nm, while Cy5 has a fluorescence emission at 670 nm.
  • Other reporter dyes include the Alexa Fluor series of dyes, which have emission wavelengths ranging from 440 nm to 805 nm.
  • Multiplex PCR is a technique used for amplification of multiple, different, nucleic acid sequences of interest in a single experiment.
  • multiplex PCR may be used to screen for the presence of nucleic acid sequences of interest from multiple, different pathogens in a single reaction, such as simultaneously screening a single sample for the presence of viral nucleic acid sequences from any of SARS-CoV, MERS, SARS-CoV-2, influenza, and Ebola viruses.
  • each pair specific to a nucleic acid sequence of interest. For example, if a sample of nucleic acid was being investigated for the presence of 10 different specific nucleic acid sequences of interest (for exam pie 10 different viruses, or 10 different genetic mutations in a patient), then at least 10 different primer pairs would be required for the multiplex PCR.
  • Beads such as microbeads (typically less than 10 pm in diameter) are commercially available and are used to conveniently capture, isolate and manipulate nucleic acids in a microfluidic environment.
  • Microbeads are typically formed from glass, polystyrene or other inert polymer, and contain surface modifications such as thiol groups, amine groups or carboxylate groups for covalent attachment of a capture moiety.
  • Microbeads can be magnetic, thus enabling transport of the microbead and associated nucleic acid material via use of one or more magnets.
  • Examples of magnetic micro beads include the Dynabead® range of products.
  • a nucleic acid of interest By covalently attaching an oligonucleotide primer to a bead, a nucleic acid of interest can be pre-hybridised to the primer and thereby bound to the bead, enabling a simple sample purification before the bead is transported through a PCR system using magnets (in the case of a magnetic bead), gravity or centrifugation.
  • Different beads can be functionalized with different primers, thus facilitating a multiplex PCR.
  • beads While this use of beads enables efficient sample purification, and localization within a PCR thermocycling chamber, the beads can cause problems during a PCR amplification process, as the beads may aggregate together, reducing access to the primer and target nucleic acid bound to the surface of the beads.
  • the present inventors have sought to develop a PCR system that enables convenient sample manipulation alongside efficient and rapid thermocycling to provide sensitive and rapid readouts.
  • the present inventors have found that it is possible to use orthogonally cleavable linkers to attach PCR reagents to solid phase beads for convenient sample manipulation, with cleavage of the linkers under controlled conditions allowing solution reactions to take place.
  • the present inventors have found that it is possible to use photocleavable and thermocleavable linkers to attach PCR primers and enzymes to beads, to confine the beads to predetermined locations before using light and heat to cleave the linkers, releasing the primers and enzyme into solution at those predetermined locations in a thermocycling chamber.
  • photocleavable and thermocleavable linkers to attach PCR primers and enzymes to beads, to confine the beads to predetermined locations before using light and heat to cleave the linkers, releasing the primers and enzyme into solution at those predetermined locations in a thermocycling chamber.
  • thermocycling results in real-time quantitative RT-PCR in a quick and sensitive process.
  • a PCR kit-of-parts comprising: a first oligonucleotide bound to a bead by a cleavable linker; a second oligonucleotide bound to a bead by a cleavable linker; and an enzyme bound to a bead by a cleavable linker; wherein the first and second oligonucleotide form an oligonucleotide pair complementary to a nucleic acid of interest and the enzyme is capable of extending nucleic acid strands; and each cleavable linker is independently selected from a photocleavable linker and a thermally cleavable linker.
  • a method of performing PCR comprising: introducing into a thermocycling chamber of a PCR system: a sample suspected of containing a nucleic acid of interest; an oligonucleotide pair complementary to the nucleic acid of interest; and an enzyme capable of extending nucleic acid strands, wherein each oligonucleotide of the oligonucleotide pair and the enzyme are independently bound by a photocleavable linker or a thermally cleavable linker to a bead dispersed in a liquid medium in the thermocycling chamber; irradiating the thermocycling chamber with light of a wavelength sufficient to cleave the photocleavable linker and/or providing heat to the thermocycling chamber sufficient to cleave the thermally cleavable linker to release the oligonucleotide pair and the enzyme into the liquid medium; subjecting the liquid medium to conditions suitable for amplification by polymerase chain
  • thermocycling chamber comprising a heater; a light source configured to emit light of a wavelength less than 400 nm to the thermocycling chamber; a light source configured to emit light of a wavelength greater than 450 nm to the thermocycling chamber; and an optical sensor configured to obtain optical signals from the thermocycling chamber.
  • a PCR kit-of-parts comprising: a first oligonucleotide bound to a bead by a cleavable linker; a second oligonucleotide bound to a bead by a cleavable linker; and an enzyme bound to a bead by a cleavable linker; wherein the first and second oligonucleotide form an oligonucleotide pair complementary to a nucleic acid of interest and the enzyme is capable of extending nucleic acid strands; and each cleavable linker is independently selected from a photocleavable linker and a thermally cleavable linker.
  • the first oligonucleotide, the second oligonucleotide and the enzyme may be bound to the same bead.
  • the first oligonucleotide and the enzyme may be bound to the same bead and the second oligonucleotide may be bound to a different bead.
  • the first oligonucleotide and the second oligonucleotide may be bound to the same bead (e.g. a first bead) and the enzyme may be bound to a different bead (e.g. a second bead).
  • Figure 1 illustrates a kit-of-parts according to the present disclosure, with each oligonucleotide and the enzyme being bound to different beads.
  • the kit 100 comprises three beads, 102a, 102b and 102c. Bound to each bead is a linker (104a, 104b and 104c respectively), which are independently photocleavable linkers or thermally cleavable linkers.
  • Linker 104a binds a first oligonucleotide 106 to bead 102a, while linker 104b binds a second oligonucleotide 108 to bead 104a, with oligonucleotides 106 and 108 forming an oligonucleotide pair complementary to a nucleic acid of interest, thus making them suitable as primers in a PCR amplification of the nucleic acid of interest.
  • enzyme 110 which is capable of extending nucleic acid strands, for example a polymerase, is bound to bead 102c by linker 104c.
  • Figure 1 illustrates oligonucleotides 106 and 108, and enzyme 110 on different beads, it will be appreciated that a single bead could be functionalized with all of oligonucleotides 106 and 108, and enzyme 110.
  • the bead or beads may be monodisperse microbeads, such as Thermo Scientific’s Opti-LinkTM Carboxylate-Modified Particles, and Dynabeads®. Regardless of whether all of the first and second oligonucleotide and the enzyme are bound to the same bead or different beads, each bead may be provided with a surface modification or functionalization to allow covalent or non-covalent coupling of the first and second oligonucleotide and the enzyme to the bead.
  • Typical surface modifications that lend themselves to covalent coupling include carboxylate modifications, amine modifications, and pre-activated surface modifications such as a tosylate modification, with the tosylate group functioning as a leaving group to an incoming nucleophile.
  • Carboxylate functionalities can be converted to corresponding esters and amides using activated ester chemistry techniques, in which a carboxylate is activated using an activating compound such as the carbodiimide N,N’-dicyclehexylcarbodiimide (DCC) with DMAP, or the related carbodiimide compound 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC, ED AC or EDCI), which is then reacted with a primary amine to form the corresponding amide, or an alcohol to form the corresponding ester.
  • DCC carbodiimide N,N’-dicyclehexylcarbodiimide
  • EDC 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • An amine surface chemistry on a bead can be converted to an amide using the same chemistry, but with the carboxylate being on the group to be coupled to the bead.
  • Surface thiol modifications can also be used, enabling coupling to, for example, a thiol- containing oligonucleotide via disulfide bond formation, while a chloromethyl functionality on the surface of a bead can react with a nucleophile, for example with a hydroxyl anion formed by deprotonation by a base of hydroxyl group).
  • the first and second oligonucleotide and the enzyme are coupled or bound to the bead using a cleavable linker selected from a photocleavable linker and a thermocleavable linker.
  • Photocleavable linkers or spacers can be used to covalently couple a molecule to a solid surface or other molecule, while allowing for selective or specific cleavage at a later stage using a wavelength of light which is selective or specific for the photocleavable moiety.
  • photocleavable linkers include o-nitro benzyl linkers (in particular o-nitrobenzyloxy and o-nitrobenzylamino linkers); a-substituted o-nitro benzyl linkers, o- nitroveratryl linkers, phenacyl linkers, p-alkoxyphenacyl linkers, benzoin linkers and pivaloyl linkers.
  • the photocleavable linker may comprise a moiety selected from an o- nitrobenzyloxy moiety; an o-nitrobenzylamino moiety; an a-substituted o-nitro benzyl moiety, an o-nitro veratryl moiety, a phenacyl moiety, a p-alkoxyphenacyl moiety, a benzoin moiety and a pivaloyl moiety
  • photocleavable linker ensures that the harsh cleavage conditions that would be required to cleave chemically cleavable linkers (acid/base chemistry or strong nucleophiles) that could cause DNA or RNA degradation can be avoided.
  • Photocleavable linkers can be cleaved with specific wavelengths of light, with a cleavage mechanism for an o-nitrobenzyl linker shown in Figure 2.
  • photocleavable moiety is shown in Table 1 in close proximity to the bead, it will be appreciated that a spacer, such as an alkyl spacer (for example a C 6 -Ci 0 alkyl spacer), or a PEG spacer may be incorporated to distance the photocleavable linker and coupled oligonucleotide/enzyme from the surface of the bead.
  • a spacer such as an alkyl spacer (for example a C 6 -Ci 0 alkyl spacer), or a PEG spacer may be incorporated to distance the photocleavable linker and coupled oligonucleotide/enzyme from the surface of the bead.
  • Methods of coupling such photocleavable linkers to activated beads include chemistries such as those mentioned above.
  • coupling of an oligonucleotide to a solid support can be achieved via the secondary hydroxyl group of the 3’-end, by first protecting the 5’-hydroxyl group and then coupling via the 3’-hydroxyl group, as is done for solid phase synthesis of oligonucleotides.
  • first oligonucleotide and the second oligonucleotide may be bound to a bead by a photocleavable linker, for example an o-nitrobenzyl linker.
  • first oligonucleotide is bound to a first bead by a first photocleavable linker
  • the second oligonucleotide is bound to a second bead by a second photocleavable linker, with the first photocleavable linker and the second photocleavable linker being cleaved under different or orthogonal cleavage conditions such as different wavelengths of light.
  • the site of binding may conveniently be the N-terminus or the C-terminus of the enzyme, or an internal lysine residue, for example.
  • amines and acids can be activated to form amide bonds.
  • the enzyme will be cleaved from the bead during photolysis, it will retain or regain its active conformation after cleavage.
  • Suitable enzymes include the thermostable polymerases Taq, Bst and Pfu.
  • any of the first oligonucleotide, the second oligonucleotide and the enzyme may be bound to a bead by a thermally cleavable linker.
  • the thermally cleavable linker may be an antibody, an affibody or an aptamer, specific to the moiety being bound (i.e. an oligonucleotide or the enzyme).
  • Affibodies are small proteins that have been engineered to bind to a target, in the same way as an antibody, and are known an antibody mimetics. Affibodies are typically much smaller than antibodies, typically comprising only a few alpha helices, but are still capable of folding into a three-dimensional structure that can be engineered to specifically bind to a target.
  • Aptamers are single stranded nucleic acids (DNA or RNA) that fold into a three- dimensional shape that can specifically bind to a target molecule analogously to antibodies, that is by recognizing and binding the three-dimensional, tertiary structure of the target rather than a complementarity binding based on Watson-Crick or Hoogsteen base-pairing.
  • Methods of generating antibodies and aptamers to protein targets and nucleic acid targets are known, and include hybridoma technology (for antibody generation) and SELEX procedures (“Systematic Evolution of Ligands by Exponential enrichment” for aptamer generation).
  • Antibodies, affibodies and aptamers are able to function as thermally cleavable linkers in that there is no covalent bond formed between the target molecule and the antibody, affibody or aptamer, with association/binding relying on non-co valent van derWaal’s forces which can be easily overcome with heating to denature or dissociate the binding.
  • thermally cleavable linker that may be utilized in the kit and methods of the present disclosure includes a binding agent specific to an enzyme, more particularly a DNA polymerase such as Taq DNA polymerase, as is used in Hot Start DNA processes.
  • Hot Start technology utilizes binding agents such as antibodies or affibodies to bind to the active site of a polymerase at room temperature, thereby blocking enzyme activity. Since the binding is non-covalent, an increase in temperature, such as the initial denaturing step of a PCR reaction, is sufficient to break the non-covalent bonds between enzyme and binding agent and restore enzyme activity.
  • the enzyme can be released and its activity restored at temperatures of about 60 °C or above for as little as one minute.
  • antibody-based Hot Start processes include the DreamTaq Hot Start DNA polymerase, the Platinum II Taq DNA polymerase and the Platinum SuperFi II DNA polymerase.
  • affibody- based Hot Start processes include the Phire Hot Start II DNA polymerase, and the Phusion Hot Start II DNA polymerase.
  • Using a thermally cleavable linker can reduce amplification products from primer dimerization and other non-specific amplification in the same way as non-bead based Hot Start processes.
  • antibodies, affibodies and aptamers can be coupled to a solid support (e.g. a bead) using approaches described above, with the option of including an inert chain spacer, such as an alkyl spacer (for example a C 6 -Cio alkyl spacer), or a PEG spacer, to ensure that the bead does not interfere with the conformational folding of the antibody, affibody or aptamer.
  • an inert chain spacer such as an alkyl spacer (for example a C 6 -Cio alkyl spacer), or a PEG spacer
  • the kit-of-parts comprises a plurality of first and second oligonucleotides forming a plurality of oligonucleotide pairs, with each oligonucleotide pair being complementary to a different nucleic acid of interest with each oligonucleotide of each oligonucleotide pair being bound to a bead by a cleavable linker.
  • each oligonucleotide pair can be considered as a primer pair for PCR amplification of a nucleic acid of interest.
  • Each oligonucleotide pair may be bound to a bead by the same type of cleavable linker, for example a nitrobenzyl linker, with different oligonucleotide pairs being bound to their respective beads by different cleavable linkers.
  • each oligonucleotide of a first oligonucleotide pair may be bound to a bead (whether the same bead or different beads) using a nitrobenzyl linker
  • each oligonucleotide of a second oligonucleotide pair may be bound to a bead (whether the same bead or different beads) using a pivaloyl linker.
  • the first pair of oligonucleotide primers is first released and an amplification of the target nucleic acid of interest attempted. If the target did not amplify, the second pair of oligonucleotides may be released and another amplification attempted; and so on until the amplification is successful.
  • a high selectivity between the primer sets is achieved due to the fact that the bulk reaction (in solution) is typically orders of magnitude more efficient than surface reactions.
  • linkers can be selectively cleaved using different wavelengths of light, as set out in Table 1 , it becomes possible to selectively cleave only one oligonucleotide pair at a time, to determine whether or not the nucleic acid of interest corresponding to that oligonucleotide pair is present in a sample.
  • the selectivity between the primer sets is achieved due to the fact that the bulk reaction is typically orders of magnitude more efficient than surface reactions.
  • the above described beads functionalised with oligonucleotides serving as PCR primers, and a DNA elongation enzyme such as a polymerase provide convenient means of delivering these PCR reagents to a thermocycling chamber. Accordingly, these beads can be considered as reagent delivery beads or reagent beads.
  • the kit-of-parts further includes a capture bead, to capture a nucleic acid of interest from a sample during purification of a sample prior to amplification.
  • an oligonucleotide of an oligonucleotide pair serving as PCR primers may serve to capture the nucleic acid of interest
  • other capturing moieties include chitosan-modified beads or silica particles.
  • capture beads and reagent delivery beads manufacturing and flexibility of the kit-of-parts and system can be improved, as purification of a target nucleic acid can be performed before any reagent beads are mixed with the target nucleic acid.
  • Capture beads comprising silica, chitosan, alumina and other type of surface chemistry can accumulate target DNA during a sample preparation phase and mixed with reagent delivery beads in a thermocycling chamber of a PCR system.
  • the system undergoes the thermal and/or photo activation processes, whereby the target DNA is released from the surface of the capture beads and the oligonucleotides and polymerase are released from the surface of the reagent delivery beads, enabling PCR reaction in the vicinity of the beads as is described below in connection with the method of the present disclosure.
  • the kit-of-parts may further comprise a reverse transcriptase bound to a bead by a photocleavable or thermally cleavable linker.
  • a kit-of-parts including a bead-bound reverse transcriptase enables the reverse transcription of an RNA sample of interest into cDNA prior to PCR amplification.
  • the reverse transcriptase and oligonucleotide pairs can be bound to beads by photocleavable linkers, while a DNA polymerase is bound to a bead by a photocleavable linker.
  • the kit- of-parts may comprise a capture bead functionalised with a poly dT oligonucleotide, to capture an RNA molecule having a poly A tail.
  • the kit-of-parts may also include a reporter molecule, such as a fluorescent dye as described herein.
  • a reporter molecule such as a fluorescent dye as described herein.
  • the kit-of-parts may comprise a non-specific fluorescent dye, such as SYBR Green I, or a target-specific fluorescent reporter molecule, such as a TaqMan hydrolysis probe.
  • the reporter molecule may be linked to an oligonucleotide of the oligonucleotide pair, such as in the Scorpion® system.
  • the reporter molecule is a target-specific fluorescent reporter molecule comprising an oligonucleotide complementary to a target region of the nucleic acid of interest, with the oligonucleotide of the reporter molecule also being bound to a bead such as described above.
  • Figure 3 illustrates this alternative kit-of-parts (also denoted generally at 100), in which a fourth bead 102d is included.
  • Bead 102d is attached to a fourth linker 104d (again, a photocleavable or a thermally cleavable linker), which binds a reporter probe 112 to bead 104d.
  • Probe 112 comprises a target-specific fluorescent reporter molecule comprising an oligonucleotide complementary to a target region of the nucleic acid of interest, which is functionalised with the fluorophore 114 and a quencher 116.
  • the kit-of-parts may further comprise one or more of dNTPs, MgCI 2 as an enzyme co-factor (although other co-factors, such as MgS0 may be used with certain enzymes), all dissolved in an aqueous buffer.
  • thermocycling chamber comprising a heater; a light source configured to emit light of a wavelength less than 400 nm to the thermocycling chamber; a light source configured to emit light of a wavelength greater than 450 nm to the thermocycling chamber; and an optical sensor configured to obtain optical signals from the thermocycling chamber.
  • thermocycling chamber comprising a heating element; a light source configured to emit light of a wavelength sufficient to cleave chemical bonds; a light source configured to emit light of a wavelength suitable to excite a fluorophore; and an optical sensor configured to obtain optical signals from the thermocycling chamber.
  • the PCR system may comprise a single chamber or a plurality of chambers provided on a substrate.
  • a plurality of thermocycling chambers may be independently operable.
  • each thermocycling chamber of a plurality of thermocycling chambers may have its own dedicated heater or heaters, so as to be independently controllable. In this way, synchronous or asynchronous control of a plurality of different PCR assays can be performed.
  • Asynchronous control of individual thermocycling chambers within a plurality of thermocycling chambers enables the amplification of a plurality of different samples using different thermocycling, or even isothermal, protocols.
  • each thermocycling chamber is provided with a plurality of independently operable heaters or heating elements, thus enabling a plurality of different PCR assays to be performed in a single thermocycling chamber, as described in the methods of the present disclosure.
  • thermocycling chamber is present as part of a microfluidic network, in which one or more microfluidic channels and chambers are fluidically connected to the thermocycling chamber, and through which a sample for purification, amplification and analysis is transported to the thermocycling chamber.
  • the thermocycling chamber may be provided in or as part of a microfluidic device.
  • the PCR system for example a microfluidic device of the PCR system, comprises a plurality of therm ocycling chambers, each fluidly connected to a central flow channel via which reagents and/or samples for amplification may be provided.
  • the PCR system for example a microfluidic device of the PCR system, comprises a plurality of thermocycling chambers, each fluidly connected to its own dedicated flow channel via which reagents and/or samples for amplification may be provided.
  • the thermocycling chamber is fluidically isolated from any other thermocycling chamber that may be present.
  • the PCR system for example a microfluidic device of the PCR system may comprise a sample purification chamber in fluid connection with the thermocycling chamber.
  • the sample purification chamber provides a location within the device to purify and isolate a nucleic acid from, for example, a cell lysis protocol prior to transporting the nucleic acid to the thermocycling chamber using a bead to which the nucleic acid can reversibly bind.
  • each thermocycling chamber may be provided with a dedicated sample purification chamber upstream from the thermocycling chamber.
  • a single sample purification chamber may serve a plurality of thermocycling chambers.
  • FIG. 4 shows an example of a microfluidic device 400 of a PCR system, incorporating a plurality of thermocycling chambers 402.
  • four thermocycling chambers 402 are approximately circular in shape (top row), with the device being provided with four elongate sample purification chambers 404 (bottom row), all provided in a cover layer 406 on a substrate 408, though it will be appreciated that other arrangements of chambers are possible.
  • each thermocycling chamber 402 and each sample purification chamber 402 is fluidically connected to a central flow channel 410, served by an inlet port 412 in cover layer 406, though it will be appreciated that this is only one example and that other configurations are possible.
  • each thermocycling chamber 402 is also fluidically connected to a common vent 414, also in cover layer 410, though it will be appreciated that each chamber may have its own dedicated vent.
  • a PCR system may comprise a substrate 408 on which the thermocycling chamber 402 is provided or the plurality of thermocycling chambers are provided.
  • the PCR system comprises a substrate on which the heater and thermocycling chamber are provided.
  • Some example substrates may include polypropylene substrates, polycarbonate substrates, polydimethylsiloxane (PDMS) substrates, silicon-based substrates, glass-based substrates, gallium arsenide based substrates, cyclic-olefin copolymer substrates (COC substrates) and/or other such suitable types of substrates for microfabricated devices and structures.
  • the substrate may be formed from a photoresist material such as SU8, an epoxy-based photoresist material, in which a pattern has been etched and into which at least a portion of the electrode assembly is to be incorporated.
  • the substrate is formed from a transparent material, thereby permitting optical detection from underneath the thermocycling chamber.
  • Thermocycling chamber 402 may be defined by or provided in cover layer 406 disposed on a substrate 408.
  • Cover layer 406 may also be termed a fluidic layer or fluidic stack, and may be formed by selectively etching or machining away regions of material so as to form a reaction chamber.
  • Cover layer 406 may comprise any material or combination of materials suitable for use in microfluidic devices, including polycarbonate, and cyclic olefin copolymer.
  • the terms “cover layer”, “microfluidic layer”, “microfluidic stack”, “fluidic layer” or “fluidic stack” refer to the components of the microfluidic device through which one or more fluids can pass during use of the microfluidic device, for example through one or more microfluidic channels and chambers. The terms are intended to encompass multiple flow paths, for example in different levels of the layer/stack, and distinguish these flow channel-containing components from other operational modules such as electronic circuitry and sensors. [00085] Other layers present in a fluidic stack may include layers of adhesive (for example pressure-sensitive adhesives) to bond the fluidic layer to the substrate and/or bond layers of a fluid stack to each other.
  • adhesive for example pressure-sensitive adhesives
  • Suitable adhesives include pressure-sensitive adhesives, which typically comprise an elastomer based on acrylic, silicone or rubber optionally compounded with a tackifier such as a rosin ester.
  • pressure sensitive adhesives are in the form of double-sided films or tape, such as the acrylic adhesives 200MP and 7956MP available from 3MTM.
  • the cover layer is provided with one or more fluid inlets and outlets, such as inlet port 412 and common vent 414.
  • fluid inlets and outlets such as inlet port 412 and common vent 414.
  • the heater or heater element is provided on or within a substrate, to provide heat to the thermocycling chamber.
  • the heater may be provided on a surface of the substrate.
  • the heater comprises a flat panel heater or one or more thermally conductive printed electrical traces or wires.
  • the substrate may comprise or is a printed circuit board (PCB), and so in some examples is termed a PCB substrate, and the heater comprises one or more printed electrical traces on a substrate to provide heat to the thermocycling chamber.
  • the heater is provided above or below the plane of the microfluidic device, or embedded into a substrate on which the thermocycling chamber is disposed.
  • the heater may comprise a Peltier device, a flat panel heater in the form of a solid-state active heat pump.
  • the heater may comprise at least one micro-heater in thermal contact with a predetermined location of the thermocycling chamber.
  • the at least one-micro-heater may comprise one or more electrical wire on a surface of the thermocycling chamber.
  • the heater receives electrical power from electrically conductive wires provided on or to the substrate to form an electrical circuit which supplies electrical current to the heater.
  • Such components may be controlled by a controller located on or off the substrate via control signals.
  • the flat panel heater or the electrical traces or wires may be formed from any suitable material used for producing microelectronics for microfluidic applications.
  • the heater (whether a flat panel heater, printed electrical trace or wire) may be formed from a metal such as platinum, aluminium, titanium, chromium, nickel, copper, silver or gold or a combination thereof.
  • the heater may be formed by sputtering or inkjet printing directly onto a substrate, or onto an adhesive layer covering the substrate.
  • Stainless steel and carbon electrodes may also be used, for example as laser cut or die punched stainless steel foils.
  • the heater comprises a titanium adhesion layer onto which a conductive gold film is deposited.
  • a flat panel heater or printed electrical trace may have a thickness in the region of 500 nm or less, for example 400 nm or less, for example 300 nm or less, for example 200 nm or less, for example about 100 nm.
  • the heater is in direct thermal contact with a liquid medium to be heated in the thermocycling chamber.
  • the thickness of the heater then correlates with V(D-t), where D is the thermal diffusivity of the liquid medium containing the beads and unbound primers, enzyme and nucleic acid of interest (in the order of 1.5 x1 O 7 ) m 2 /s) and t is the heating duration.
  • D the thermal diffusivity of the liquid medium containing the beads and unbound primers
  • enzyme and nucleic acid of interest in the order of 1.5 x1 O 7 ) m 2 /s
  • t is the heating duration.
  • a passivation layer or dielectric layer can be deposited onto the heater. The dielectric layer may be spun on or sputtered onto the heater and substrate.
  • the heater may be provided with a dielectric or passivation coating of polyimide, SU-8, silicon oxide, silicon nitride, aluminium oxide, aluminium nitride or any combination / stack thereof.
  • a dielectric or passivation coating of polyimide, SU-8, silicon oxide, silicon nitride, aluminium oxide, aluminium nitride or any combination / stack thereof.
  • Kapton® Another suitable material is Kapton®, which may be incorporated into a coating or stack with any of the aforementioned materials.
  • each thermocycling chamber may have a plurality of independently operable heaters, with each heater provided at a predetermined location.
  • each thermocycling chamber can have multiple reaction zones which are independently or asynchronously controllable relative to the each other. In this way, a plurality of different PCR assays (i.e. a multiplexed PCR), each requiring a different thermocycling protocol, can be performed in a single reaction chamber.
  • thermocycling protocols may require shorter or longer annealing times, or higher or lower annealing temperatures, based on length and content of the primers used (longer oligonucleotides, or oligonucleotides having high proportions of G:C base pairs will have higher melting temperatures, which will affect annealing of the primer to the template strand).
  • the predetermined locations at which heaters are provided are spaced apart to avoid cross-contamination.
  • the individual locations are spaced from 100 pm to 1000 pm apart, in some examples from 200 to 800 mih apart, in some examples 300 to 600 pm apart, and in some examples 500 pm apart. Since the methods of the present disclosure may utilise amplification conditions which do not include fluid flow within the thermocycling chamber, spacing apart the individual heaters in this way avoids cross-contamination as amplification of nucleic acid material sufficient to obtain a positive or negative detection result is quicker than any diffusion of the nucleic acid of interest, and quicker than diffusion of oligonucleotide primers used to amplify the nucleic acid of interest.
  • the PCR system may be provided with a magnet or a plurality of magnets in or under the substrate.
  • the magnet may comprise a permanent magnet or an electromagnet.
  • the magnet or magnets can draw the beads to a predetermined location within the sample purification chamber or the thermocycling chamber and thus increase a local concentration of bead and bound oligonucleotide/enzyme.
  • the PCR system comprises a light source configured to emit light of a wavelength sufficient to cleave chemical bonds, to enable cleavage of a photocleavable linker, thereby releasing a bound oligonucleotide, enzyme or reporter molecule into solution in a liquid medium in the thermocycling chamber.
  • a light source configured to emit light of a wavelength less than 400 nm to the thermocycling chamber, for example less than 380 nm, for example less than 370 nm, for example less than 360 nm, for example from 280 nm to 360 nm.
  • the light source configured to emit light of a wavelength less than 400 nm may comprise a mercury lamp, a laser or a UV LED.
  • the light source may be configured, or operable, to emit light with a power of at least 10 mW/mm 2 , for example at least 20 mW/mm 2 , for example at least 30 mW/mm 2 , for example at least 40 mW/mm 2 , for example at least 50 mW/mm 2 , for example at least 60 mW/mm 2 , for example at least 70 mW/mm 2 , for example at least 80 mW/mm 2 , for example at least 90 mW/mm 2 , for example at least 100 mW/mm 2 , for example at least 110 mW/mm 2 , for example at least 120 mW/mm 2 , for example at least 130 mW/mm 2 , for example at least 140 mW/mm 2 , for example at least 150 mW/mm 2 , for example at least 160 mW/mm 2 , for example at least 170 mW/mm 2 , for example at least 180 mW/mm 2
  • the light source may be configured, or operable, to emit light with a power of less than 200 mW/mm 2 , for example less than 190 mW/mm 2 , for example less than 180 mW/mm 2 , for example less than 170 mW/mm 2 , for example less than 160 mW/mm 2 , for example less than 150 mW/mm 2 , for example less than 140 mW/mm 2 , for example less than 130 mW/mm 2 , for example less than 120 mW/mm 2 , for example less than 110 mW/mm 2 , for example less than 100 mW/mm 2 , for example less than 90 mW/mm 2 , for example less than 80 mW/mm 2 , for example less than 70 mW/mm 2 , for example less than 60 mW/mm 2 , for example less than 50 mW/mm 2 , for example less than 40 mW/mm 2 , for example less than 30 mW/mm 2
  • the light source may be configured, or operable, to emit light with a power of from 10 to 200 mW/mm 2 , for example from 20 to 190 mW/mm 2 , for example from 30 to 180 mW/mm 2 , for example from 40 to 170 mW/mm 2 , for example from 50 to 160 mW/mm 2 , for example from 70 to 150 mW/mm 2 , for example from 75 to 140 mW/mm 2 , for example from 80 to 130 mW/mm 2 , for example from 85 to 120 mW/mm 2 , for example from 90 to 110 mW/mm 2 , for example about 100 mW/mm 2 .
  • the light source may be configured, or operable, to emit light for any of the above described power values for a period of at least 1 second, for example at least 2 seconds, for example at least 3 seconds, for example at least 4 seconds, for example at least 5 seconds, for example at least 6 seconds, for example at least 7 seconds, for example at least 8 seconds, for example at least 9 seconds, for example at least 10 seconds, for example at least 11 seconds, for example at least 12 seconds, for example at least 13 seconds, for example at least 14 seconds, for example at least 15 seconds, for example at least 16 seconds, for example at least 17 seconds, for example at least 18 seconds, for example at least 19 seconds, for example at least 20 seconds.
  • the light source may be configured, or operable, to emit light for any of the above described power values for a period of less than 20 seconds, for example less than 19 seconds, for example less than 18 seconds, for example less than 17 seconds, for example less than 16 seconds, for example less than 15 seconds, for example less than 14 seconds, for example less than 13 seconds, for example less than 12 seconds, for example less than 11 seconds, for example less than 10 seconds, for example less than 9 seconds, for example less than 8 seconds, for example less than 7 seconds, for example less than 6 seconds, for example less than 5 seconds, for example less than 4 seconds, for example less than 3 seconds, for example less than 2 seconds, for example about 1 second.
  • the light source may be configured, or operable, to emit light for any of the above described power values for a period of from 1 to 20 seconds, for example from 2 to 19 seconds, for example from 3 to 18 seconds, for example from 4 to 17 seconds, for example from 5 to 16 seconds, for example from 6 to 15 seconds, for example from 7 to
  • the light source may be configured, or operable, to emit light with a power of from 10 to 200 mW/mm 2 , for example from 20 to 190 mW/mm 2 , for example from 30 to 180 mW/mm 2 , for example from 40 to 170 mW/mm 2 , for example from 50 to 160 mW/mm 2 , for example from 70 to 150 mW/mm 2 , for example from 75 to 140 mW/mm 2 , for example from 80 to 130 mW/mm 2 , for example from 85 to 120 mW/mm 2 , for example from 90 to 110 mW/mm 2 , for example about 100 mW/mm 2 for a period of from 1 to 20 seconds, for example from 2 to 19 seconds, for example from 3 to 18 seconds, for example from 4 to 17 seconds, for example from 5 to 16 seconds, for example from 6 to
  • 15 seconds for example from 7 to 14 seconds, for example from 8 to 13 seconds, for example from 9 to 12 seconds, for example about 10 seconds,
  • the light source may be configured, or operable, to emit light having an overall power density of from 1 mJ/cm 2 to 200 mJ/cm 2 , for example 1 mJ/cm 2 to 180 mJ/cm 2 , for example 1 mJ/cm 2 to 170 mJ/cm 2 , for example 1 mJ/cm 2 to 160 mJ/cm 2 , for example 10m J/cm 2 to 150 mJ/cm 2 , for example 15mJ/cm 2 to 140 mJ/cm 2 , for example 20mJ/cm 2 to 130 mJ/cm 2 , for example 25mJ/cm 2 to 140 mJ/cm 2 , for example 50 mJ/cm 2 to 120 mJ/cm 2 , for example 75mJ/cm 2 to 110 mJ/cm 2 for example 80m J/cm 2 to 100 mJ/cm 2 .
  • the PCR system comprises a light source configured to emit light of a wavelength sufficient to cleave a photocleavable linker as described herein, for example light of a wavelength greater than 450 nm; and an optical sensor configured to obtain optical signals from the therm ocycling chamber.
  • the optical sensor is a fluorescence sensor and the optical signals are fluorescence signals.
  • fluorescent molecules are used as reporter molecules in PCR amplification, with the fluorescence intensity proportional to the amount of amplified nucleic acid material. Wavelengths of light suitable to cause fluorescence of reporter molecules used in PCR amplification are generally greater than 450 nm, with the wavelength of fluorescence being even greater.
  • the light source comprises a laser diode, or an LED, configured to emit light of a wavelength suitable to cause fluorescence of a fluorescent reporter molecule during a PCR amplification process.
  • a wavelength suitable to cause fluorescence of a fluorescent reporter molecule during a PCR amplification process For example, SYBR Green I, absorbs blue light with a A max of 497 nm, and emits green light with a A max of 520 nm.
  • the detector may be a charge coupled device (CCD) or pin photodiode configured to detect the emitted fluorescent light.
  • the optical sensor comprises a detector, which may be a charge coupled device (CCD) or pin photodiode to detect the emitted fluorescent light.
  • the optical sensor is arranged above or below the thermocycling chamber, for example above or below a plane in which the liquid sample is being thermocycled.
  • a microfluidic device on which the thermocycling chamber is provided comprises an optical window or opening that allows transmission of light therethrough to an optical sensor located in the PCR system but external to the microfluidic device, or within the microfluidic device itself.
  • the optical sensor is embedded into a cover of the microfluidic device.
  • the thermocycling chamber may be provided on or in a cassette, or chip, to be used in the PCR system.
  • the microfluidic device may be a single use or disposable device.
  • the microfluidic device may be configured to be inserted into or received by a port in the system.
  • the microfluidic device may be provided with one or more fluidic connections that are configured to engage with one or more corresponding fluidic connections in the apparatus, to enable fluid flow from the system into the microfluidic device, for example to enable transfer of a sample injected into an injection port of the system to be transferred to a sample purification chamber or a thermocycling chamber of the microfluidic device.
  • the or each chamber of the microfluidic device may be filled with sample prior to inserting the microfluidic device into the system, for example by manual pipetting a sample solution through an inlet port such as a Luer connector or membrane valve.
  • the PCR system may comprise an electrical interface, configured to contact an electrical interface provided on the device.
  • the electrical interface on the system may be coupled to any component of the device that requires electrical current to operate. Examples of such components include the heater elements, either in flat panel form or printed conductive trace form, and actuators for controlling fluid flow within the device, or for transporting reagent or sample beads from one chamber to another.
  • the electrical interfaces may be multi-pin input/output off board connecters, for example 44-pin connectors that enable electrical coupling of the device to a computer module of the PCR system.
  • Each pin of the electrical interface may provide an electrical contact to a specific component of the device, such as the flat panel heater or micro-heaters described herein.
  • the electrical coupling of the device to the system allows control signals from the computer module to be sent to the PCR device so that electrical current can be sent to desired modules of the device.
  • the PCR system may comprise a computer control module.
  • the computer control module comprises a processor comprising hardware architecture to retrieve executable code from a data storage device or computer-readable medium and execute instructions in the form of the executable code.
  • the processor may include a number of processor cores, an application specific integrated circuit (ASIC), field programmable gate array (FPGA) or other hardware structure to perform the functions disclosed herein.
  • the executable code may, when executed by the processor, cause the processor to implement the functionality of one or more hardware components of the device and/or system such as one or more heater assemblies and/or one or more optical detectors.
  • the processor may receive input from and provide output to a number of the hardware components, directly or indirectly.
  • the computer control module may communicate with such components via a communication interface which may comprise electrical contact pads, electrical sockets, electrical pins or other interface structures. In one example, the communication interface may facilitate wireless communication.
  • the computer control module facilitates the introduction of a sample into a sample purification chamber and a thermocycling chamber, or into multiple thermocycling chambers.
  • the computer control module may control a series of valves and pumps in the system oron the device to direct flow of a test sample or solution to the thermocycling chamber after purification.
  • the computer control module may further control the processing of a sample in a thermocycling chamber, for example by subjecting the thermocycling chamber to thermocycling conditions.
  • the computer control module may control, through the output of control signals, the operation of one or more heater elements to control the temperature and duration of heating within the or each thermocycling chamber.
  • the computer control module may control, through the output of control signals the provision of one or more pulses of electrical energy to the heater to generate waves of thermal energy in a liquid medium within the thermocycling chamber, radiating from the surface adjacent the heater into the liquid medium.
  • the computer control module may also control, through the output of control signals, the operation of a light source to control emission of light of a wavelength less than 400 nm to the thermocycling chamber to cleave a photocleavable linker.
  • the computer control module may also control, through the output of control signals, the operation of a light source to control emission of light of a wavelength greater than 450 nm to the thermocycling chamber to monitor progress of an amplification reaction via excitation of a fluorophore with the light of a wavelength greater than 450 nm.
  • the computer control module may control, through the output of control signals, the operation of the optical sensor, to monitor progress of the PCR amplification via detection of the excited fluorophore and thereby detection of the presence of a nucleic acid of interest in a sample.
  • a sample and reagents may undergo various selected reactions, various selected heating cycles and various sensing operations under the control of the computer control module.
  • a method of manufacturing a PCR system comprising: forming a thermocycling chamber provided with a heater; and configuring: a light source to emit light of a wavelength less than 400 nm to the thermocycling chamber; a light source to emit light of a wavelength greater than 450 nm to the thermocycling chamber; and an optical sensor to obtain optical signals from the thermocycling chamber.
  • thermocycling chamber provided with a heating element; and configuring: a light source configured to emit light of a wavelength sufficient to cleave chemical bonds; a light source configured to emit light of a wavelength suitable to excite a fluorophore; and an optical sensor configured to obtain optical signals from the thermocycling chamber.
  • the heater may be in the form of a flat panel heater disposed on a substrate as described previously.
  • the substrate may be etched to provide one or more recesses or traces into which the heater can be inserted or printed.
  • thermocycling chamber may comprise forming or providing a cover layer having one or more thermocycling chambers, and arranging the cover layer on the substrate so as to align a thermocycling chamber with at least one heater on the substrate.
  • the upper surface of the substrate forms the bottom surface of the thermocycling chamber.
  • the thermocycling chamber has an internal diameter of from 5 to 200 pm, for example from 5 to 100 pm, for example from 10 to 100 pm.
  • the methods comprise configuring: a light source configured to emit light of a wavelength sufficient to cleave chemical bonds; a light source configured to emit light of a wavelength suitable to excite a fluorophore.
  • the methods comprise configuring: a light source to emit light of a wavelength less than 400 nm to the thermocycling chamber, and a light source to emit light of a wavelength greater than 450 nm to the thermocycling chamber.
  • the light sources may be independently selected from mercury lamps, LEDs and lasers.
  • the light sources may be directly integrated into the thermocycling chamber, for example into a wall or cover of the thermocycling chamber or they may be located elsewhere in the system but configured to emit light of the appropriate wavelengths to the thermocycling chamber.
  • a single light source is provided, which is configured to emit light of different wavelengths at different times.
  • the single light source may be operable to emit light of a wavelength sufficient to cleave chemical bonds (for example light of a wavelength less than 400 nm) in order to cleave one or more photocleavable linkers to release PCR reagents into a liquid medium, prior to a second wavelength being emitted with the second wavelength being suitable to excite a fluorophore during amplification.
  • a wavelength sufficient to cleave chemical bonds for example light of a wavelength less than 400 nm
  • the use of one or more bandpass filters enables operation of a single light source in this manner.
  • the method comprises configuring an optical sensor to obtain optical signals from the thermocycling chamber.
  • the optical sensor is a fluorescent sensor.
  • the optical sensor is directly integrated into the thermocycling chamber, for example into a wall or cover of the thermocycling chamber or is located elsewhere in the system but configured to receive signals from the thermocycling chamber.
  • thermocycling chamber of a PCR system a sample suspected of containing a nucleic acid of interest; an oligonucleotide pair complementary to the nucleic acid of interest; and an enzyme capable of extending nucleic acid strands, wherein each oligonucleotide of the oligonucleotide pair and the enzyme are independently bound by a photocleavable linker or a thermally cleavable linker to a bead dispersed in a liquid medium in the thermocycling chamber; irradiating the thermocycling chamber with light of a wavelength sufficient to cleave the photocleavable linker and/or providing heat to the thermocycling chamber sufficient to cleave the thermally cleavable linker to release the oligonucleotide pair and the enzyme into the liquid medium; subjecting the liquid medium to conditions suitable for amplification by polymerase chain reaction;
  • the PCR system may be as described herein.
  • Figure 5 illustrates the basic principle of the method. In Figure 5, all components which are common to Figures 1 and 3 have not been labelled, for simplicity.
  • a nucleic acid of interest labelled 118
  • a kit of parts comprising first and second oligonucleotide primers and an enzyme, each bound to a different bead by a photocleavable or thermally cleavable linker.
  • Figure 5 shows nucleic acid 118 as being in solution, it may also be bound to a nucleic acid capture bead as described previously.
  • nucleic acid 118 may be p re- mixed with the kit-of- parts to hybridise it to an oligonucleotide primer before being introduced into a thermocycling chamber.
  • nucleic acid 118 hybridises to an oligonucleotide primer, thus bringing it into close proximity with the PCR reagents. As noted, this may occur in a sample purification chamber or in the thermocycling chamber.
  • Figure 5 schematically shows a reporter probe being added to the system. Again, this may be introduced into a thermocycling chamber as part of a kit-of- parts, bound to a bead, or it may be introduced separately to the kit-of-parts, for example dissolved in a liquid medium introduced into the thermocycling chamber.
  • Figure 5 also indicates that the action of light and/or heat cleaves the photocleavable/thermally cleavable linkers, to release the oligonucleotide primers and enzyme into solution.
  • a complex of the target nucleic acid, the oligonucleotide primers, the reporter probe and the enzyme forms in solution in the liquid medium in the thermocycling chamber, and amplification by PCR can occur - in solution - leading to generation of copies 118’ of the original target nucleic acid of interest.
  • a sample suspected of containing a nucleic acid of interest may be introduced into the thermocycling chamber in a liquid medium.
  • the sample may comprise a nucleic acid for analysis, e.g. a nucleic acid of interest, which is to be amplified in a method as described herein.
  • the sample may comprise a plurality of nucleic acids for analysis which are to be amplified in a method as described herein.
  • the sample is suspected of comprising one or a plurality of nucleic acid sequences of interest.
  • the sample is obtained from one or more of a blood sample, a tissue sample, a saliva sample or mucosal sample.
  • the nucleic acid sample is obtained using a swab.
  • the nucleic acid sample is isolated from the bodily fluid or tissue via which it was obtained. That is, the sample suspected of containing a nucleic acid of interest may be purified in a purification chamber with the purified nucleic acid of interest being introduced into the thermocycling chamber.
  • the nucleic acid sample is not isolated from the bodily fluid or tissue via which it was obtained.
  • the nucleic acid sample obtained from a subject may be incorporated into a test solution with or without any isolation or preparation.
  • the nucleic acid sample obtained from a subject is dissolved or dispersed in an aqueous solution, thus forming a test solution.
  • the liquid medium introduced into the thermocycling chamber may comprise an aqueous solution of a nucleic acid of interest.
  • the nucleic acid sample may be bound to a capture bead as described herein with the capture bead being dispersed or suspended in the liquid medium.
  • An oligonucleotide pair complementary to the nucleic acid of interest and an enzyme capable of extending nucleic acid strands may also be present in a liquid medium containing the nucleic acid of interest introduced into the thermocycling chamber, or may be introduced into the thermocycling chamber independently of the nucleic acid of interest. In either case, each oligonucleotide of the oligonucleotide pair and the enzyme are independently bound by a photocleavable linker or a thermally cleavable linker to a bead as described herein.
  • the sample suspected of containing a nucleic acid of interest can be pre-mixed with one or more beads to which are bound at least one oligonucleotide of the oligonucleotide pair prior to being introduced into the thermocycling chamber.
  • the nucleic acid of interest can in effect be isolated from the rest of a biological sample through prehybridisation to a PCR primer before being introduced into the thermocycling chamber.
  • Reagent beads and/or samples for amplification may be transported into a purification chamber, when used, and into the thermocycling chamber via injection pressure and capillary forces, or through the use of magnets (when the beads are magnetic), gravity or centrifugation.
  • the beads are mixed with a crude sample suspected of containing a target nucleic acid, which may take place in a sample purification chamber of the PCR system.
  • the target nucleic acid attaches to the beads’ nucleic acid attachment moiety (for example on a capture bead, or an oligonucleotide primer bound to a bead).
  • the beads are then removed from the crude sample solution and transferred to a wash buffer.
  • the wash buffer promotes removal of any species from the bead surface that may interfere with the later amplification.
  • the liquid medium may further comprise dNTPs and an enzyme co-factor such as MgCI 2 .
  • the liquid medium which may also be termed a test solution, comprises the four standard dNTPs, i.e. dGTP, dCTP, dATP and TTP and may also contain one or more reporter molecules that permit monitoring of the amplification by optical means as described herein. However, in some situations the reporter molecule is bound to a bead as described.
  • a liquid medium comprising at least the four standard dNTPs, i.e. dGTP, dCTP, dATP and TTP, and any necessary enzyme cofactor may be present in the thermocycling chamber prior to the reagent beads and sample of nucleic acid being introduced.
  • the test solution comprises a plurality of oligonucleotide or primer pairs, each pair complementary to a different nucleic acid of interest and each oligonucleotide of each pair bound to a bead as described.
  • a multiplexed PCR analysis is enabled.
  • a portion of oil is provided into the thermocycling chamber.
  • the function of the oil is to prevent evaporation of any liquid from the liquid medium during amplification.
  • light mineral oils such as a white oil are provided.
  • the volume of oil provided is in excess of the volume of the liquid medium. In some examples, sufficient oil is provided to completely envelop the liquid medium.
  • the method of performing PCR may include a step of confining each oligonucleotide of the oligonucleotide pair and the enzyme to a predetermined location within the thermocycling chamber prior to being released from the bead or their respective beads. This may be accomplished through the use of magnetic beads, and a series of magnets arranged within the PCR system, to manipulate the bead or beads so as to transport them to and within the thermocycling chamber. In this way, the bead or beads can be brought not only into close proximity with a heater or heater element, but also in close proximity to one another resulting in high local concentrations of the reagents at the predetermined location or locations.
  • the PCR reagents - now in solution - are in high concentrations in close proximity to the heater. This not only increases the sensitivity of the assay and the speed of amplification (due to the solution reactions occurring faster than a corresponding reaction on a solid support), but also enables a more rapid thermocycling as only that small volume of the liquid medium needs to be thermocycled, with the rest of the liquid medium acting as a heat sink to provide cooling to the small volume being thermocycled as is described later.
  • the liquid medium has a volume of less than 100 pl_, for example less than 50 pl_, for example less than 25 mI_, for example less than 10 mI_, for example about 5 mI_. In some examples, the liquid medium has a volume of greater than 5 mI_, for example greater than 10 mI_, for example greater than 25 mI_, for example greater than 50 mI_, for example about 100 mI_.
  • the liquid medium at the predetermined location adjacent the heater may be a small percentage (less than 1 %) of the entire liquid medium in the thermocycling chamber.
  • the method of performing PCR includes a step of irradiating the thermocycling chamber with light of a wavelength sufficient to cleave the photocleavable linker.
  • This step may comprise irradiating with light of a wavelength less than 400 nm.
  • photocleavable linkers undergo photolysis at wavelengths of less than 400 nm, i.e. in the UV region of the spectrum.
  • This step may comprise irradiating with UV-A light, i.e. light having a wavelength from 315-400 nm.
  • thermocycling chamber with light having a wavelength within these ranges is sufficient to cleave, for example, a nitrobenzyl photocleavable linker to release a PCR reagent in the form of an oligonucleotide primer, or a DNA elongating enzyme into the liquid medium.
  • a suitable light source for performing the irradiation may be a mercury lamp or a UV LED. Suitable conditions have been described previously.
  • Oligonucleotide primers bound to beads via photocleavable linkers may not be released all at once, but can in fact be titrated in during the course of the amplification process. This can be achieved by controlling the UV flux. Ways of controlling UV flux include controlling exposure time, using neutral density filters in front of the light source, and by controlling the current of the light source (e.g. a UV LED). This controls the primer concentration in the reaction, especially in the beginning, and helps avoid primer dimerization and so non-specific amplification products.
  • Ways of controlling UV flux include controlling exposure time, using neutral density filters in front of the light source, and by controlling the current of the light source (e.g. a UV LED). This controls the primer concentration in the reaction, especially in the beginning, and helps avoid primer dimerization and so non-specific amplification products.
  • the method may also include providing heat to the thermocycling chamber sufficient to cleave a thermally cleavable linker.
  • a heater in thermal contact with the thermocycling chamber may provide the heat necessary to cleave a thermally cleavable linker.
  • a thermally cleavable linker may be an antibody, an affibody or an aptamer which specifically but non-covalently binds an oligonucleotide primer, ora DNA elongating enzyme.
  • the heat necessary to cleave a thermally cleavable linker may be accomplished by heating the thermocycling chamber comprising the liquid medium to a temperature of at least 50 °C, for example at least 60 °C, for example at least 70 °C, for example at least 80 °C, for example at least 90 °C, for example up to about 95 °C. Heating the thermocycling chamber comprising the liquid medium to one of these temperatures may be performed as part of a first denaturation step of a PCR amplification thermocycle, or it may be performed prior to this step.
  • the subsequent cleavage and amplification conditions may be performed in the absence of any fluid flow within the thermocycling chamber. That is, once the beads have been localized to a predetermined location in close proximity to the heater associated with the thermocycling chamber, all fluid flow whether by stirring, external injection pressure, capillary pressure or gravity is ceased. As a result, once the oligonucleotide and enzyme reagents have been cleaved from the bead or their respective beads, these species only slowly diffuse from the bead surface to a localized region near the beads, and the rate of diffusion is greater than the rate of amplification. Thus, when the liquid medium is subjected to amplification conditions, only that localized region needs to be thermocycled.
  • the concentration of the nucleic acid of interest to be amplified is, at the start of the method, is greater than zero, for example greater than 10 23 M (mo I/I), for example greater than 10 21 M, for example greater than 10 2 ° M, for example greater than 10 19 M. in these amounts, the amplification is sufficiently sensitive to produce an amount of amplification products that can be suitably detected.
  • the concentration of the nucleic acid of interest to be amplified in the PCR may be less than 1 nM, for example less than 30 pM, for example less than 1 pM, for example less than 0.1 pM, for example less than 10 fM, for example less than 1 fM, for example less than 0.1 fM.
  • the number of molecules of the nucleic acid of interest to be amplified in the method is, at the start ofthe method, preferably less than 500,000, particularly preferably less than 200,000, particularly preferably less than 100,000, particularly preferably less than 10,000. In this way, it is possible to prevent the amplification reaching saturation before its end.
  • the nucleic acid of interest may be an RNA, and the method may further comprise using the RNA as a template for a reverse transcriptase to produce a cDNA strand which can then be used in a PCR amplification process.
  • the reverse transcriptase may also be bound to a bead using a photocleavable or thermally cleavable linker along with any necessary RNA binding primer.
  • the liquid medium in the thermocycling chamber may be heated to a temperature in the region of 40-50 °C to allow reverse transcription to occur and produce the cDNA in situ which can then be used directly in a PCR amplification upon further heating of the liquid medium to about 95 °C to cleave a thermally cleavable linker binding a DNA polymerase to a bead.
  • the liquid medium is subjected to conditions suitable for amplification by polymerase chain reaction.
  • the conditions suitable for amplification by polymerase chain reaction may comprise thermocycling in the absence of fluid flow within the thermocycling chamber.
  • the conditions suitable for amplification by polymerase chain reaction may comprise heating the predetermined location to a denaturing temperature of the nucleic acid of interest by providing a pulse of energy to a micro-heater adjacent the predetermined location.
  • the conditions suitable for amplification by polymerase chain reaction may comprise pulse-controlled amplification, in which a time varying heat flux is applied to the liquid medium, resulting in a time varying temperature gradient in the liquid medium, as will be explained in more detail below.
  • General amplification conditions comprise cycling a PCR mixture between three different temperatures or ranges of temperatures: typically 94-98 °C for denaturation; 50-65 °C for annealing, and 70-80 °C for chain extension or elongation, depending on polymerase.
  • each thermocycle necessarily also involves a heating step which begins before the denaturing step, but which may also overlap with the denaturing step.
  • the temperature of the liquid medium with respect to the starting temperature or the temperature of a previous elongation step is increased at least locally, i.e. at the predetermined locations where the beads and released reagents are concentrated, in order to facilitate denaturing.
  • Subjecting the liquid medium to conditions suitable for amplification by polymerase chain reaction may therefore comprise heating the liquid medium at the predetermined location to a denaturing temperature of the nucleic acid of interest by providing a pulse of energy to a micro-heater adjacent the predetermined location.
  • a localised temperature of at least 90° C for example at least 95° C, can rapidly be reached.
  • the length of time of heating is the total duration, in which the heater transmits heat to the predetermined location with a power suitable for denaturing of a nucleic acid sample at that predetermined location, which may correspond to a transient, localised heating of the liquid medium to a temperature of at least 90° C. If therefore the heating is produced, for example, by a flat panel heater, the heating time is the total duration, in which heat flows from the flat panel heater to the predetermined location to bring about a temperature increase at that location that is suitable for denaturing.
  • the duration of heating to effect denaturation may be less than 10 seconds, for example less than 5 seconds, for example less than 3 second, for example less than 1 second, for example less than 500 ms (milliseconds), for example less than 250 ms, for example less than 100 ms, for example less than 50 ms, for example less than 25 ms, for example less than 10 ms, for example less than 8 ms, for example less 3 ms, for example less than 1 ms, for example less than 500 ps (microseconds), for example less 300 ps, for example less than 100 ps, for example less than 50 ps, for example less than 30 ps, for example less than 10 ps.
  • the heater is able to rapidly heat a small interfacial volume of liquid medium in the thermocycling chamber and rapidly and reliably effect denaturation.
  • a cooling step begins before the annealing step, in order to reach the temperature required for annealing, usually 50- 65 °C. If the temperature in the previous denaturing step was only locally increased, the cooling may take place through heat diffusion in the liquid medium, once power to the heater has been stopped. Since the rest of the volume of the liquid medium is in comparison to the heated volume much larger, the rest of the volume serves as a heat sink to rapidly cool the heated area.
  • the duration of cooling to effect annealing of the primers to the nucleic acid at the predetermined location may be less than 10 seconds, for example less than 5 seconds, for example less than 3 second, for example less than 1 second, for example less than 500 ms (milliseconds), for example less than 250 ms, for example less than 100 ms, for example less than 50 ms, for example less than 25 ms, for example less than 10 ms, for example less than 8 ms, for example less 3 ms, for example less than 1 ms, for example less than 500 ps (microseconds), for example less than 300 ps, for example less than 100 ps, for example less than 50 ps, for example less than 30 ps, for example less than 10 ps.
  • a heating step begins before the chain extension or elongation step, in order to reach the temperature required for chain extension, usually 70-80 °C.
  • the length of time of heating for chain extension is the total duration, in which the heater transmits heat to the predetermined location with a power suitable for chain extension of a nucleic acid sample at that predetermined location, which may correspond to a transient, localised heating of the liquid medium to a temperature of about 70-80 °C. Ifthe heating is produced, for example, by a flat panel heater, the heating time is the total duration, in which heat flows from the flat panel heater to the predetermined location to bring about a temperature increase at that location suitable for chain extension.
  • the annealing temperature is equal to the chain extension temperature. If the annealing temperature is equal to the chain extension temperature, only one temperature cycle with two different temperatures is required to amplify the nucleic acid of interest.
  • the melt temperatures of the primers and the polymerase used may be selected so that at the primer melting temperature the polymerase used can still synthesize DNA at a sufficient speed.
  • the temperature for annealing and chain extension is achieved by global heating and the denaturing step is achieved through localised heating at the predetermined location.
  • the duration of heating to effect chain extension may be less than 10 seconds, for example less than 5 seconds, for example less than 3 second, for example less than 1 second, for example less than 500 ms (milliseconds), for example less than 250 ms, for example less than 100 ms, for example less than 50 ms, for example less than 25 ms, for example less than 10 ms, for example less than 8 ms, for example less 3 ms, for example less than 1 ms, for example less than 500 ps (microseconds), for example less 300 ps, for example less than 100 ps, for example less than 50 ps, for example less than 30 ps, for example less than 10 ps.
  • the heater comprises an array of 75 gold-coated tungsten wires (15 pm diameter, 200 nm Au coating) arranged on an internal surface of the thermocycling chamber.
  • Localised heating of a layer of liquid medium of a few micrometers depth adjacent the wire is achieved via application of sub-millisecond voltage pulses to the wires.
  • a pulse at substantial peak power in the order of 1 kW for less than 500 microseconds is applied to the wire array from a 10 mF capacitator loaded to 30-40 V via a MOSFET (metal-oxide-semiconductor field-effect transistor; serving as a fast switch). This is sufficient to locally heat the solution at the predetermined location to greater than 90 °C to denature the amplicons in solution.
  • MOSFET metal-oxide-semiconductor field-effect transistor
  • localized heating for a denaturing step can be achieved by heating a thin film flat panel heater (tungsten film, 15 pm thickness with a, 200 nm thick Au coating) with a heat flux of 4000kW/m 2 for 1 ms (millisecond), with a heat flux of 400kW/m 2 for 10 ms (milliseconds), or with a heat flux of 40 kW/m 2 for 100 ms (milliseconds) from a 10 mF capacitator loaded to 30-40 V via a MOSFET (metal-oxide- semiconductor field-effect transistor.
  • MOSFET metal-oxide- semiconductor field-effect transistor
  • the heater is able to rapidly heat a small interfacial volume of liquid medium in the thermocycling chamber and rapidly effect chain extension of the primers by the polymerase, using a nucleic acid of interest as the template.
  • the liquid medium within the thermocycling chamber is heated globally by a second heater, and maintained at a temperature of 50-60 °C (i.e., a temperature suitable for annealing).
  • a second heater may be provided at a different location to the heater configured to provide localised heating.
  • the second heater may be provided in a cover layer of the system, in thermal contact with the liquid medium in the thermocycling chamber.
  • thermocycling chamber for example micro-heaters
  • cooling of the micro-heaters is almost instantaneous when power to the heater is switched off.
  • Localised heating is much more rapid than heating of the whole liquid medium as less energy needs to be transferred, and a bulk reaction (i.e., reaction in solution) is much more rapid than a surface-based reaction. Therefore, a more rapid and energy efficient PCR method can be realised.
  • PCR amplification is typically monitored by fluorescence of a reporter molecule adapted for that purpose.
  • the methods of the present disclosure may further include introducing a fluorophore into the thermocycling chamber, and detecting an optical signal by irradiating the thermocycling chamber with light of a wavelength greater than 450 nm.
  • An optical sensor is configured to obtain optical signals from the therm ocycling chamber.
  • the optical sensor for example a fluorescence sensor, is used to detect and measure the fluorescence level.
  • the fluorescence sensor detects and measures the fluorescence level after each thermocycle, or after 5 thermocycles, or after 10 thermocycles, or any number of cycles as required.
  • a nucleic acid of interest is present in the sample, it will be amplified through the thermocycling, using the complementary oligonucleotide primer pair. Since the amplification of that particular nucleic acid of interest takes place in the thermocycling chamber, measurement of any presence or increase in fluorescence is an indication that the nucleic acid of interest was present in the sample or test solution. The sooner that a positive result (via fluorescence detection) confirms that a nucleic acid of interest is present in a test solution, the quicker the overall test time.
  • the present disclosure thus enables a simple, rapid point-of-care diagnostic that can accurately and simultaneously screen for nucleic acid sequences of interest.
  • kits-of-parts, methods, systems and related aspects have been described with reference to certain examples, it will be appreciated that various modifications, changes, omissions, and substitutions can be made without departing from the spirit of the disclosure. It is intended, therefore, that kits-of-parts, methods, systems and related aspects be limited only by the scope of the following claims. Unless otherwise stated, the features of any dependent claim can be combined with the features of any of the other dependent claims, and any other independent claim.

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Abstract

Kit de pièces pour PCR. Le kit de pièces comprend les éléments suivants : un premier oligonucléotide lié à une bille par un lieur clivable ; un second oligonucléotide lié à une bille par un lieur clivable ; et une enzyme liée à une bille par un lieur clivable ; les premier et second oligonucléotides formant une paire d'oligonucléotides complémentaire d'un acide nucléique d'intérêt et l'enzyme étant capable d'étendre des brins d'acide nucléique ; et chaque lieur clivable étant indépendamment choisi parmi un lieur photoclivable et un lieur thermoclivable. L'invention concerne également un procédé de réalisation de la PCR et un système de PCR.
PCT/US2021/043118 2021-07-26 2021-07-26 Kit de pièces, procédé et système pour pcr WO2023009099A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARYA MANIT ET AL.: "Basic principles of real-time quantitative PCR", EXPRT REW. MOL. DIAGN., vol. 5, no. 2, 2005, pages 209 - 219, XP008180265, DOI: 10.1586/14737159.5.2.209 *
HAUSCH F , JÄSCHKE A: "Multifunctional DNA conjugates for the in vitro selection of new catalysts.", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 28, no. 8, 15 April 2000 (2000-04-15), GB , pages E35 - E35-3, XP002575759, ISSN: 1362-4962 *
KOCH T, ET AL.: "PHOTOCHEMICAL IMMOBILIZATION OF ANTHRAQUINONE CONJUGATED OLIGONUCLEOTIDES AND PCR AMPLICONS ON SOLID SURFACES", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 11, no. 04, 1 January 2000 (2000-01-01), US , pages 474 - 483, XP002944640, ISSN: 1043-1802, DOI: 10.1021/bc000011h *
NILSSON J. ET AL.: "Heat-mediated activation of affinity-immobilized Taq DNA polymerase", BIOTECHNIQUES, vol. 22, April 1997 (1997-04-01), pages 744 - 751, XP008107641 *

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